A toxin of Clostridiun difficile is a cause of postantibiotic pseudomembranous colitis. Some antibiotics provide a selective niche for the growth and consequent toxin production. The morbidity of this condition is high and its seriousness makes the availability of a specific sensitive diagnostic test desirable. In a more general sense, elucidation of the mode of action of this toxin will provide a basis for understanding the disease and of the use of the toxin as a probe in cellular biology. The first objective of this study was to purify toxin. This toxin aggregates in the presence of carbohydrate-like materials (e.g., filtration gels) which make it behave like a high molecular weight molecule, ca. 500,000, whereas pure toxin, isolated from density gradients, has an apparent molecular weight of 50,000, determined by analytical ultracentrifugation. The toxin also binds to polyacrylamide and to agarose gels used to monitor it. Recently we have found that two toxin subfractions separated by ion exchange chromatography differ in carbohydrate content and cytotoxicity in tissue culture and in lethality in animals. Gel analysis showed that the protein band pattern is also different. We are presently investigating these subfractions of toxin. Our studies indicate the toxin may be a lectin which is altered in biological activity when it binds carbohydrate. Immunization of animals with toxin from carbohydrate rich medium has not resulted in neutralizing antibody but much precipitating antibody. Using enzyme linked immune assay we have shown, in collaboration, that specific goat anti-rabbit globulin combines with this toxin but will not neutralize it. We are now using low carbohydrate toxin for immunization for neutralizing antibody. Our new knowledge of the physico-chemical characteristics of C. difficile toxin will allow us to proceed more rapidly with its molecular description and action and with the development of a practical method for its clinical detection.